FOLIA BIOLOGICA ET GEOLOGICA 61/1, 75–87, LJUBLJANA 2020 UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS AND PROVIDE HEALTH BENEFIT FOR HUMANS UV ABSORBIRAJOČE SNOVI V AJDI ŠČITIJO RASTLINE IN PRISPEVAJO K ZDRAVJU LJUDI Lea LUKŠIČ 1 , Aleksandra GOLOB 2,3 , Maria MRAVIK 4 & Mateja GERM 2,5 http://dx.doi.org/10.3986/fbg0070 ABSTRACT UV absorbing compounds in buckwheat protect plants and provide health benefit for humans Buckwheat became a pan-Eurasian crop, when it expand- ed via Himalaya to Europe. Common buckwheat is one of the oldest domesticated crops in Asia, while Tartary buckwheat is still thriving as a wild or weedy plant. Buckwheat belongs to dicotyledonous crops that can tolerate poor soils and extreme environment conditions. Buckwheat grows on high elevation, where the intensities of UV radiation are usually high. Buck- wheat is a fast-growing plant rich in flavonoids, which absorb UV radiation and have an antioxidant potential. Flavnoids have positive effect also on human health. Besides common buck- wheat f lour, Tartary buckwheat f lour is more and more used in preparing dishes, due to its much higher content of flavonoids rutin and quercetin compared to common buckwheat. There- fore, the studies on how the technological procedures of pre- paring Tartary buckwheat bread affect the content, availability and efficacy of f lavonoids in buckwheat bread have been made. Buckwheat is commonly used in the dishes in Japan (soba noo- dles), China (buckwheat noodles), Korea (buckwheat noodles), Italy (buckwheat polenta), France (galettes), Slovenia (kasha, žganci). Common buckwheat and T artary buckwheat are plants suitable for designing foods with good functional value and healthy features. Therefore, it has been determined that differ- ent technological procedures, such as hydrothermal treatment of grain, sourdough fermentation, dough preparation and bak- ing influences the availability and changes in the content of f lavonoids, rutin and quercetin and antioxidant activity in sour bread and food products, made with buckwheat flour. Key words: Common buckwheat, Tartary buckwheat, sourdough bread, rutin, quercetin, flavonoids, UV absorbing compounds IZVLEČEK UV absorbirajoče snovi v ajdi ščitijo rastline in prispevajo k zdravju ljudi Ajda je postala vseevrazijska kultura, ko se je preko območja Himalaje razširila v Evropo. Navadna ajda je ena najstarejših gojenih rastlin v Aziji, medtem ko tatarska ajda še vedno uspeva tudi kot divja ali plevelna rastlina. Ajda spada med gojene dvokaličnice, ki lahko prenašajo slaba tla in eks- tremne razmere v okolju. Ajda raste na visoki nadmorski višini, kjer je intenziteta ultravijoličnega sevanja običajno vi- soka. Ajda je hitro rastoča rastlina, bogata z flavonoidi, ki ab- sorbirajo UV sevanje in imajo antioksidativni potencial. Fla- vonoidi pozitivno vplivajo tudi na zdravje ljudi. Poleg moke iz navadne ajde se moka iz tatarske ajde vse pogosteje uporablja pri pripravi jedi, ker ima v primerjavi z navadno ajdo veliko večjo vsebnost flavonoidov kot sta rutin in kvercetin. Zato so bile narejene študije o tem, kako tehnološki postopki priprave kruha iz tatarske ajde vplivajo na vsebnost, razpoložljivost in učinkovitost flavonoidov v ajdovem kruhu. Ajdo je zelo po- gosto uporabljajo v jedeh na Japonskem, na Kitajskem, Koreji, v Italiji, v Franciji, Sloveniji. Navadna ajda in tatarska ajda, sta rastlini primerni za pripravo živil z dobro funkcijsko vred- nostjo in lastnostmi ugodnimi za zdravje. Ugotovljeno je bilo, da različni tehnološki postopki, kot so hidrotermična obdela- va zrnja, mlečnokislinska fermentacija, priprava testa in peka, vplivajo na dostopnost in spremembe v vsebnosti f lavonoidov, rutina in kvercetina in antioksidativno aktivnost kislih kruhov in prehranskih izdelkov pripravljenih iz ajdove moke . Ključne besede: navadna ajda, tatarska ajda, kruhi s kis- lim testom, rutin, kvercetin, flavonoidi, UV absorbirajoče snovi 1 Zavod Nutridharma – Košenice 92, SI-8000 Novo mesto, Slovenija, e-mail: nutridharma@gmail.com 2 Univerza v Ljubljani, Biotehniška fakulteta, oddelek za biologijo - Večna pot 111, SI-1000 Ljubljana, Slovenija 3 e-mail: Aleksandra.Golob@bf.uni-lj.si 4 Slovak University of Agriculture in Nitra, Faculty of biotechnology and food sciences – Tr. A. Hlinku 2, 949 76 Nitra, Slovakia e-mail: xmravik@is.uniag.sk 5 e-mail: mateja.germ@bf.uni-lj.si LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 76 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 Buckwheat origins in southern China, probably Y unnan province (Ohnishi 1998), from where it gradually spread to the north of China, and further on across Rus- sia and Ukraine (Kreft 1995). It became a pan-Eurasian crop, when it expanded via the Himalaya region to Eu- rope (Hunt et al. 2018). Common buckwheat is one of the oldest domesticated crops in Asia, while Tartary buckwheat is still thriving as a wild or weedy plant (Tsuji & Ohnishi 2009). Buckwheat is an ancient di- cotyledonous crop that tolerates poor soils and extreme environments (Bilal Pirzadah et al. 2013). Buckwheat, as a robust and undemanding plant, is becoming an im- portant alternative staple foods crop. The residual nutri- ents from preceding crops are often sufficient for its ad- equate growth. Tartary buckwheat is grown in Luxem- bourg, and as a mixed crop with common buckwheat in Bosnia and Herzegovina. Tartary buckwheat has been introduced also to Slovenia, Italy and Sweden (Gao et al. 2016). Crop production of grains that are botanically not cereals, such as buckwheat (Fagopyrum spp.), is increas- ing (Ačanski et al. 2015). It is a fast-growing plant rich in flavonoids, which contribute efficiently to its biologi- cal activity (Horbowicz et al. 2011). Buckwheat grows on high elevation, where the intensities of UV radiation 1 INTRODUCTION are usually high. Thus, it is feasible to study the effect of UV radiation on the synthesis of UV absorbing com- pounds. Among grain crops, research on buckwheat is raising attention because of its content of many healthy compounds (Christa & Soral-Śmietana 2008; Chi- tarrini et al. 2014). The comparative study of Sofic et al. (2010) showed that out of 50 medical plant species, rue (Ruta graveolens) plants contained the highest amount of rutin (86.6 mg/g DM) followed by buckwheat flowers (53.5 mg/g DM) (Budzynska et al. 2018). Besides com- mon buckwheat flour, Tartary buckwheat flour is more and more used in preparing dishes, due to its much high- er content of rutin and quercetin compared to common buckwheat (Fabjan et al. 2003; Jiang et al. 2007; Kreft 2016). Therefore, the studies on how the technological procedures of preparing Tartary buckwheat bread affect the content, availability and efficacy of flavonoids in buckwheat bread have been made (Vogrinčič et a l. 2010; Zhang et al. 2010; K o č e va r G l avač et al. 2017; Costan - tini et al. 2014; Lukšič et al. 2016a, 2016b). Buckwheat is commonly used in the dishes in Japan (soba noodles), China (buckwheat noodles), Korea (buckwheat noodles), Italy (buckwheat polenta), France (galettes), Slovenia (kasha, žganci) (Škrabanja et al. 2018). 2 THE EFFECT OF UV RADIATION ON THE CONTENT OF UV ABSORBING COMPOUNDS Flavonoids have great potential to scavenge reactive oxy gen species (ROS), compounds, which are also pro- duced during water shortage (Hideg & Strid 2017). The production of UV-absorbing compounds in plants like flavonoids and related phenyl-propanoids are primary protective mechanisms protecting plants from poten- tially damaging solar UV radiation (Zhang et al. 2012; Barnes et al. 2016, Liang et al. 2006). Hideg & Strid (2017) reported that flavonoids can scavenge reactive oxygen species (ROS) by acting as antioxidants. The production is determined by the UV dose, radiation quality and time of exposure. Since UV-B triggers the secondary metabolic pathway, it may serve as a signifi- cant stimulator for plant antioxidant activity (Sebas - tian et al. 2018). Kishore et al. (2010) evidenced the positive correlations between phenolic compounds and the amounts of certain antioxidants and altitude of the growing site of Tartary buckwheat. In buckwheat leaves rutin and rutin-oxidaze enhance the defence against UV radiation, low temperature and water shortage (Su- zuki et al. 2015). In the study of Gaberščik et al. (2002) common buckwheat plants (Fagopyrum esculentum Moench, variety ‘Darja’) were grown in outdoor experi- ments under reduced and ambient UV-B levels, and an UV-B level simulating 17% ozone depletion in Ljubljana (Slovenia). UV-B radiation induced synthesis of UV ab- sorbing compounds. The flavonoid synthesis is signifi- cantly enhanced by UV radiation as shown in many studies (Gaberščik et al. 2002; Suzuki et al. 2005; Golob et al. 2018). Regvar et al. (2012) studied the ef- fects of increased UV-B radiation that simulates 17% ozone depletion on fungal colonisation and concentra- tions of rutin, catechin and quercetin in common buck- wheat and Tartary buckwheat. They found out induc- tion of shoot quercetin concentrations in UV-B-treated common buckwheat plants, but no differences in flavonoid concentrations in Tartary buckwheat. Tartary buckwheat had higher concentrations of flavonoids comparing to common buckwheat. Authors presumed that concentrations of these secondary metabolites are the result of genetic pre-adaptation of Tartary buck- wheat to higher altitudes, where they protect against UV LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 77 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 radiation. Jovanovi ć et al. (2006) studied the behaviour of the enzymatic antioxidant defense system in common buckwheat leaves and seedlings subjected to enhanced UV-B radiation (supplemented UV-B light (radiation 290-320 nm) for 90 min by use of a UV-B lamp (HPQ 100 W Phillips). Plants received 49 kJ m -2 biologically effective UV-B radiation. UV-B applied treatment caused enhanced level of methanol-soluble flavonoids, in line with studies from Gaberščik et al. (2002) and Suzuki et al. (2005). The study with fifteen populations of Tartary buckwheat from different elevations exposed to elevated UV -B radiation showed that the sensitivity of plants to UV-B radiation is not only associated with the ambient UV-B level in natural habitats but also with the relative growth rate of genotype (Yao et al. 2007). Thus, future effort to breed for more tolerant cultivars is pos- sible. Yao et al. (2008) studied the effects of enhanced UV -B radiation on crop growth, morphology, reproduc- tion, and physiology in three cultivars of Fagopyrum es- culentum originating from different altitudes and re- vealed that enhanced UV-B radiation significantly af- fected plant growth, development and production, a cultivar originating from Qinghai-Tibet plateau being the most tolerant. Debski et al. (2016) studied the impact of short- term UV-B treatment on the content of flavonoids and photosynthetic pigments in cotyledons, and the growth of common buckwheat seedlings. Seedlings were sub- jected to different doses of UV-B, 5 W∙m –2 and 10 W∙m – 2 . Exposure to UV -B enhanced the amount of anthocya- nins in cotyledons while inhibiting hypocotyl elonga- tion, but had no effect on the content of photosynthetic pigments. Exposure to UV-B radiation did not affect rutin levels or cause a decrease in it with respect to dif- ferent cultivars. Anthocyanin type and their contents in Tartary buckwheat stems were investigated by Eguchi and Sato (2009). The ratio of each anthocyanin type to total an- thocyanins varied with nodal positions in an outdoor experiment. This experiment showed that UV stress in- fluences the ratio of specific anthocyanins to total an- thocyanins. This growth chamber experiment showed that the ratio of cyanidin-3-O-rutinoside to total antho- cyanins was higher under UV conditions in comparison to non-UV conditions. Authors presume that Tartary buckwheat may accumulate cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside systematically to protect plants against UV stress. In the experiments of Yao et al. (2006), Tartary buckwheat was grown in field plots under near-ambient solar UV-B (approximately 84–88% of solar UV-B), at- tenuated solar UV-B radiation (43–49% reduction in solar UV -B), and supplemental UV -B radiation (two lev- els: 5.30 and 8.50 kJ m-2 day-1). The amount of photo- synthetic pigments was lowered by the ambient and en- hanced UV -B radiation, while the UV -B absorbing com- pounds and rutin concentration increased, except at the highest level of UV -B irradiance exposure. Authors con- cluded that Tartary buckwheat is a potentially UV-B sensitive species, and also, that the crop response to UV-B radiation is associated with UV-B intensity, envi- ronmental factors and growing season. Orsak et al. (2001) provided evidence about chang- es of total polyphenols, phenolcarboxylic acids, and ascorbic acid in three buckwheat samples (seeds, seed- lings, and plants of Fagopyrum esculentum Moench, cv. Pyra and Emka, and Tartary buckwheat Fagopyrum ta- taricum Gaertner), induced by UV-C irradiation (lamb- da = 253.7 nm, P = 75 W, 0 - control, 42 and 84 h). Au- thors found out that F. tataricum contained much higher total polyphenol and rutin levels in comparison to F. esculentum, and that UV-C irradiation affected seeds causing an increase in the amounts of total polyphenols and rutin. Tsurunaga et al. (2013) studied the effects of vari- ous light compositions on the levels of anthocyanins, rutin, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radi- cal scavenging activity in common buckwheat sprouts. Sprouts were irradiated with different sources of visible and ultraviolet light. Authors examined the effect of UV-B at wavelengths of 260-320 nm, 280-320 nm, and 300-320 nm on the synthesis of flavonoid compounds. Their results showed that irradiation with UV-B>300 nm increased the levels of anthocyanins and rutin, as well as the DPPH radical scavenging activity. When sprouts were irradiated with UV-B light at wavelengths of 260-300 nm, yellowing or withering occurred. The effects of blue and UV-A (365 nm)/UV-C (254 nm), or their combinations, on the levels of total flavo- noids, rutin, quercetin, PAL, CHI, rutin degrading en- zymes (RDEs), and DPPH radical scavenging activity in Tartary buckwheat sprouts were researched (Ji et al. 2016). Authors found out that blue light in combination with UV-C (BL+UV-C) enhanced the accumulation of total flavonoids, rutin, and quercetin, while this effect was not observed when blue light was combined with UV-A (BL+UV-A). To conclude, UV radiation stimulates phenylpro- panoid biosynthetic pathway leading to the accumula- tion of compounds that protect plants from the UV caused damage. However, these responses differ regard- ing the intensity of UV radiation, buckwheat species and on elevation of origin of cultivar. Some studies showed that Tartary buckwheat is more tolerant to UV radiation since it originates in high altitudes. LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 78 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 Common buckwheat and T artary buckwheat are used in different parts of the world for making various food products. Bonafaccia et al. (2003) evidenced that the grain of both of these cultivated buckwheat species con- tains up to 27% fibre. Buckwheat seeds are considered as a prebiotic food because they can increase the lactic acid bacteria in the intestine due to their content of resistant starch (Škrabanja et al. 1998, 2001). Buckwheat has small starch granules and an amylose content of starch higher than cereals, but lower than those of legumes (Škrabanja & Kreft 1998; Schirmer et al. 2013). Buckwheat (Fagopyrum esculentum) herb is used for herbal medicinal products, for preparing green buck- wheat tea, for producing buckwheat green leaf f lour as an additive to certain food products, and the fresh green plant parts can be used as a vegetable (Kreft et al. 2006). Buckwheat contains more rutin than the majority of other grain crops, fruits, and vegetables (Li et al. 2011). Many authors reported that seeds of Tartary buckwheat have higher contents of high quality proteins and higher concentrations of flavonoids rutin and quercetin than those of common buckwheat seeds (Fabjan et al. 2003; Gao et al. 2016). Fabjan et al. (2003) reported that Tar- tary buckwheat contains about 100-fold more rutin than does common buckwheat. Buckwheat is thus an impor- tant source of anti-oxidant activity in functional foods (Holasova et al. 2002) due to the presence of the flavo- noids rutin and quercetin in buckwheat grain and prod- ucts, because of their anti-oxidant and anti-inflamma- tory effects. Rutin and quercetin were also present in baked biscuits made from flour of both species of buck- wheat (Wieslander et al. 2011). Buckwheat products decrease cholesterol levels and also improve lung capac- ity in humans (Wieslander et al. 2011; Yang et al. 2014). Extracts from common buckwheat and Tartary buckwheat can also protect DNA from damage caused by hydroxyl radicals (Vogrin čič et al. 2010). Experi- ments showed that buckwheat flour can improve diabe- tes, obesity, hypertension, hypercholesterolemia and constipation (Li & Zhang 2001). 3 IMPORTANCE OF BUCKWHEAT AS THE STAPLE CROP 4 RUTIN AND QUERCETIN TRANSFORMATION DURING PREPARATION OF BUCKWHEAT SOURDOUGH BREAD AND THE EFFECT OF HYDROTHERMAL TREATMENT OF TARTARY BUCKWHEAT GRAIN TO THE TRANSFORMATION OF RUTIN TO QUERCETIN Pseudocereals have received increased interest in recent years due to the growing awareness of the need for healthy diets. Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a pseudocereal rich in dietary beneficial components. It is a popular food source, containing bal- anced amino-acid composition of its proteins, fiber, ret- rograded starch, trace elements, vitamins and antioxi- dants, including flavonoids (Holasova et al. 2002; Bonafaccia et al. 2003; Fabjan et al. 2003; Pongrac et a l. 2016). Buckwheat does not contain gluten proteins, so it is safe for people suffering from gluten intolerance (Vogrinčič et al. 2010; Kocjan Ačko 2015). Tartary buckwheat possesses phenolic compounds, such as rutin, quercetin, kaempferol-3-rutinoside and flavanol triglycoside, and has a high antioxidant activity that helps to reduce the risk of major chronic diseases (Tian et al. 2002). Tartary buckwheat contain more rutin (a quercetin-3-rutinoside; 10 and 40 mg/g, respectively) than most vegetables, fruits and grain crops (Li & Zhang 2001), and more rutin (up to about 14.7 mg per g DM) than common buckwheat (up to about 0.1 mg per g DM) (Fabjan et al. 2003; Kreft 2016). Tartary buck- wheat was widely grown in the territory of Slovenia since the beginning of 19th century (Fabjan et al. 2003). During the 20th century, the cultivation of Tartary buckwheat gradually decreased, due to cultivation of other crops (Kreft 1995, 2011). However, common buckwheat bread was traditionally made in the past, and its use is reviving in present times. Due to much higher content of rutin and quercetin in Tartary buckwheat flour (Jiang et al. 2007; Qin et al. 2010), the Tartary buckwheat bread has been prepared to investigate the effects of hydrothermal treatment of grain, sourdough fermentation and the baking process on rutin and quercetin content and on the antioxidant activities of common buckwheat and Tartary buckwheat bread (Costantini et al. 2014; Lukšič et al. 2016a, 2016b). One of the ways to prepare bread involves sour- dough fermentation, which can be accompanied by the formation of lactic acid and acetic acid that have an im- pact on dough processing and the preparation of sour- dough bread (Michalska et al. 2008). Many authors have reported that sourdough fermentation can affect the improvement of structural and sensory properties, as well as persistence of sour bread (Gobbetti et al. 2014; Rizzello et al. 2016; Rinaldi et al. 2017; Ua A r a k LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 79 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 et al. 2017). Experiments showed that sourdough fer- mentation can improve the availability of proteins and minerals, total content of dietary fiber, total content of phenolic substances and antioxidant activity of sour bread (Boskov Hansen et al. 2002; Gandhi & Dey 2013; Costantini et al. 2014; Rizzello et al. 2016). It has been reported that Tartary buckwheat sour bread had a lower content of carbohydrates, lower glycemic index and a lower energy value than the same amount of Tartary buckwheat f lour (Novotni et al. 2012; Costan - tini et al. 2014). In sour bread, various substances such as alcohols, aldehydes, esters, hydrocarbons, ketones, terpenes, furans and phenols are also produced during the process of lactic acid fermentation (Boskov Hansen et al. 2002). Microorganisms in sourdough starter can also form some new nutritional components, such as peptides and other amino acid derivatives, and some prebiotic polysaccharides (De Vos 2005). It has been found that proteases released by yeast and the enzymes of selected lactobacilli in sourdough starter can metabo- lise gluten in wheat flour (Weiser et al. 2008). In the studies, a decrease in the content of rutin and its conversion into quercetin, which is prevented by var- ious types of heat treatment of food products made from Tartary buckwheat and common buckwheat have been reported (Vogrinčič et al. 2010). Changes in the con- tent of other substances with antioxidant activity have also been measured (Vogrinčič et al. 2010; Zhang et a l. 2010; Sakač et al. 2011 ). Cho and Lee (2015) repor ted that changes in the content of rutin and other antioxi- dants have not been affected by rapid frying of the noo- dles, while cooking caused a significant reduction in the content of rutin in the noodles prepared from wheat flour enriched with rutin extract from Tartary buck- wheat bran. Jambrec et al. (2015) reported that in full wheat noodles with the addition of pre-autoclaved (120 ° C) flour of common buckwheat, the conversion of rutin into quercetin decreased. While during the cooking of these noodles, the conversion of rutin into quercetin did not occur at all. The decrease of phenolic substances in noodles with the addition of buckwheat flour was com- parable to decrease of phenolic substances in control sample (whole grain wheat noodles). In the experiments of Qin et al. (2013) it has been found that soaking of Tar- tary buckwheat grains (40 °C, 12-14 h) influenced the reduction of the starch and rutin content and inf luenced the increase in the content of quercetin, kaempferol, iso- quercitrin, total flavonoids and phenolic substances. After the pre-soaked grains of Tartary buckwheat were treated with steam (100 ℃, 40-60 min), the content of total f lavonoids and total phenolic substances decreased, while the content of rutin in the grain samples increased. It is possible that the process of decomposition of rutin was initiated in the process of soaking grain of Tartary buckwheat, and the steam treatment process triggered a reconnection of rutin. Sensoy et al. (2006) provided evidence that shows that the processing (roasting) of buckwheat flour had no effect on the content of total phenolic substances in buckwheat flour. The DPPH method, used for determination of antioxidant activity, however, showed a slight decrease in antioxidant activi- ty of buckwheat flour while roasting at 200 °C for 10 min, while the roasting at 170 °C had no effect on the reduction of antioxidant activity. The significant de- crease in anti-oxidant activity in Tartary buckwheat flour as a result of various thermal treatments such as roasting, steam-pressure heating, and microwaving, has been reported (Zhang et al. 2010). A small decrease in the anti-oxidant activity in common buckwheat flour roasted for 10 min at 200 °C has also been noted (Yasu - da & Nakagawa 1994). The results of the experiment suggests that optimization of processing is the key to maintaining healthy substances in buckwheat products. Bread is a staple food for the majority of the world populations and contributes substantially to the intakes of certain nutrients. Common buckwheat and especially Tartary buckwheat flour possesses some phenolic com- pounds, such as rutin, quercetin and a high antioxidant activity, that is why this type of f lour is suitable to obtain bread with improved nutritional value and healthy fea- tures. Therefore, the studies have been made on how the technological procedures of preparing common buck- wheat and Tartary buckwheat bread affect the content, availability and efficacy of flavonoids in buckwheat bread. It has been proven that, in bread made with Tar- tary buckwheat flour, rutin concentration decreased, whereas the quercetin concentration increased and re- mained stable during processing (Vogrinčič et al. 2010). Lukšič et al. 2016a, 2016b reported that during Tartary buckwheat bread making there was a transfor- mation of a large portion of rutin into quercetin. Breads containing common buckwheat f lour, contained several flavonoids, such as rutin and quercetin, and had higher antioxidant activity than wheat bread (Lin et al., 2009). The combined effects of sourdough fermentation and the baking process on the flavonoid concentrations and antioxidant properties of common buckwheat and Tar- tary buckwheat sourdough starter, bread dough and sourdough bread have also been studied. It has been es- tablished that common buckwheat and Tartary buck- wheat bread making is feasible without any addition of wheat or gluten, by using the sour bread starter proce- dure (Costantini et al. 2014; Lukšič et al. 2016a, 2016b). Tartary buckwheat breads made with 100% Tartary buckwheat flour contained the highest phenol (53.3 mg GAE/g) and flavonoid (16.8 mg RE/g) contents, mean- LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 80 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 while Tartary buckwheat sour bread, containing 10% of chia (Salvia hispanica L.) flour had the highest antioxi- dant activity (32.0 mmol Fe 2+ E/g and 128.6 mmol GAE/g, respectively) compared to 100% common buck- wheat and wheat sour breads and common buckwheat and wheat sour breads fortified with 10% of chia flour (Costantini et al. 2014). On the contrary, in another experiment a higher antioxidant activity has been mea- sured in common buckwheat bread compared to Tar- tary buckwheat bread. This might be because of the syn- thesis of substances with antioxidant properties, includ- ing certain Maillard reaction products that occur in bread during thermal treatment (Zhang et al. 2010; Lukšič et al. 2016a). A similar result were established in a study of Vogrinčič et al. (2010) in which Tartary buckwheat bread and breads made of mixtures of Tar- tary buckwheat and wheat flour were studied. A de- crease in polyphenol concentration through baking was observed in all samples. The high DPPH (2,2-diphenyl- 1-picrylhydrazyl) scavenging capacity in mixed breads (32-56%) and in Tartary buckwheat bread (85-90%) de- creased slightly through the bread making process, while an increase of antioxidant activity in bread made of 100% wheat f lour during bread making was observed. In the experiments of Lukšič et al. (2016a) sourdough bread was prepared of flour of common buckwheat and of Tartary buckwheat to follow the transformation of rutin and quercetin during sourdough fermentation, bread making procedure and baking of bread. During Tartary buckwheat sourdough fermentation, there was conversion of rutin to quercetin. In the Tartary buck- wheat sourdough bread there was no rutin, whereas there was 5.0 mg/g quercetin. In common buckwheat bread, neither rutin nor quercetin were present. Vogrinčič et al. (2010) reported that with the addition of water to mixtures containing Tartary buckwheat dur- ing the preparation of the Tartary buckwheat dough made with yeast, rutin concentration decreased, while quercetin concentration increased. The rutin concentra- tion continued to decrease during the bread baking pro- cess, while the concentration of quercetin remained sta- ble. After baking, rutin (0.47 mg/g) was present only in bread made of 100% Tartary buckwheat f lour along with quercetin (4.83 mg/g). Suzuki et al. (2015) reported that when using a version of Tartary buckwheat with traces of rutinosidase, bread of a flour mixture of Tartary buckwheat and wheat with 0.63 mg/g rutin was pre- pared, representing approximately 50% of the retained rutin in bread, compared to the source material (flour). Hydrothermal treatment is a process that involves heating with hot water or steam, followed by cooling and drying of buckwheat groats to produce husked buck- wheat or kasha. It is the traditional technology known and still applied in Slovenia, Croatia, Poland, Ukraine and Russia (Kreft 2003). In a study of Lukšič et al. (2016b) the impact of hydrothermal treatment on ex- tractability of flavonoids from starchy matrix was inves- tigated. Tartary buckwheat grain was hydrothermally treated and milled to yield hydrothermally treated flour. In control sample, not hydrothermally treated Tartary buckwheat f lour, most of extractable rutin (8 mg of rutin per g DM (dry matter)) was extracted during the first 20 min of extraction. In hydrothermally treated Tartary buckwheat flour only 4 mg of rutin per g DM was ex- tracted in 20 min, and 7 mg of rutin per g DM within 8 h, respectively. This data indicates that, during the hydro- thermal treatment, rutin becomes embedded in the f lour matrix. Slowly extracted rutin was protected from trans- formation to quercetin during bread making procedure. From an initial 7 mg of extractable rutin per g DM in hydrothermally treated buckwheat flour, Tartary buck- wheat bread contained 2 mg of rutin per g DM, and 6 mg of quercetin per g DM. No other Tartary buckwheat bread making technology which would be able to con- serve such an amount of rutin from flour through the process to the final bread product have been reported. Many studies certificate that both, fermentation process and heat treatment affects changes in content and accessibility of substances with antioxidant proper- ties in buckwheat products. However, these changes dif- fer regarding buckwheat species and plant properties, preparation process, fermentation method and thermal treatment used. This information contributes to a better understanding of the effects of different food prepara- tion methods on substances with antioxidant activity and information on the persistence of rutin and querce- tin in sourdough bread and other food products. These findigs are as well important for designing foods with high concentrations of flavonoids and good functional value. POVZETEK Ajda izvira iz južne Kitajske, verjetno province Yunnan, od koder se je postopoma razširila na sever Kitajske in naprej po Rusiji in Ukrajini. Postala je vseevropska ra- stlina ko se je razširila preko Himalaje v Evropo. Nava- dna ajda je ena najstarejših gojenih rastlin v Aziji, med- tem ko tatarska ajda še vedno uspeva tudi kot divja ra- LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 81 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 stlina ali plevel. Ajda je starodavna dvokaličnica, ki prenaša revno prst in ekstremna okolja. Ajda, kot robu- stna in nezahtevna rastlina, postaja pomemben vir za pridelavo osnovnih živil. Hranilne snovi, ki ostanejo v zemlji iz gojenih rastlin prejšnjih sezon, so pogosto za- dostne za njeno ustrezno rast. Tatarsko ajdo sejejo v Luksemburgu in kot mešani pridelek z navadno ajdo v Bosni in Hercegovini. Tatarsko ajdo smo pred kratkim začeli ponovno sejati tudi v Sloveniji, Italiji in na Šved- skem. Pridelava ajde (Fagopyrum spp.), se povečuje. Je hitro rastoča rastlina, bogata z flavonoidi, ki učinkovito prispevajo k njeni biološki aktivnosti. Ajda raste na vi- soki nadmorski višini, kjer so intenzitete UV sevanja običajno visoke. Zato je smiselno preučevati učinke UV sevanja na sintezo spojin, ki absorbirajo UV . Število raz- iskav o ajdi se povečuje ker ima visoko vsebnost snovi, ki pozitivno vplivajo na zdravje ljudi. Primerjalna študija je pokazala, da je od 50 vrst ra- stlinskih zdravilnih rastlin vinska rutica vsebovala naj- večjo količino rutina (86,6 mg / g SM), sledijo ajdovi cvetovi (53,5 mg / g SM). Poleg moke iz navadne ajde, se za pripravo jedi vedno bolj uporablja tatarska ajdova moka, zaradi veliko večje vsebnosti rutina in kvercetina. Zato so izdelane študije o tem, kako tehnološki postopki priprave tatar- skega ajdovega kruha ajde vplivajo na vsebino, razpolo- žljivost in učinkovitost flavonoidov v ajdovem kruhu. Ajda se zelo pogosta uporablja za pripravo jedi na Japon- skem (soba rezanci), na Kitajskem (ajdovi rezanci), v Ko- reji (ajdovi rezanci), Italiji (ajdova polenta), Franciji (ga- lettes), Sloveniji (kaša, žganci). Zaradi naraščanja zavedanja o pomenu zdrave pre- hrane, rastline, ki botanično niso žita, med katera uvr- ščamo tudi tatarsko ajdo (Fagopyrum tataricum Gaer- tn.) ponovno vzbujajo zanimanje med ljudmi. Tatarsko ajdo odlikuje odlična hranilna vrednost, saj ima urav- noteženo aminokislinsko sestavo, vsebuje prehranske vlaknine, retrogradiran škrob, elemente v sledovih, vi- tamine in antioksidante, vključno s flavonoidi (Hola - sova et al. 2002; Bonafaccia et al. 2003; Fabjan et al. 2003; Pongrac et al. 2016). Ajda ne vsebuje glutena in je varno živilo tudi za osebe z ugotovljenimi intolerancami na gluten (Vogrinčič et al. 2010; Kocjan Ačko 2015). Tatarska ajda ima visoko antioksidativno aktivnost in vsebuje fenolne spojine, kot so rutin, kvercetin, kaemp- ferol-3-rutinozid in triglikozid f lavanol, ki lahko prispe- vajo k zmanjšanju možnosti za nastanek kroničnih bole- zni (Tian et al. 2002). Tatarska ajda vsebuje več rutina (10 in 40 mg/g), kot večina zelenjave, sadja in žit (Li & Zhang 2001) in več rutina (do 14,7 mg/g suhe mase) kot navadna ajda (do 0,1 mg/g suhe mase) (Fabjan et al. 2003; Kreft 2016). Tatarsko ajdo so na območju Slove- nije pridelovali že v začetku 19. stoletja (Fabjan et al. 2003). V obdobju 20. stoletja pa se je pridelava tatarske ajde zmanjševala, predvsem zaradi pridelave drugih žit (Kreft 1995, 2011 ). Kruh iz moke navadne ajde so tradi- cionalno pekli že v preteklosti, sedaj pa se zanimanje za ajdov kruh ponovno veča. Zaradi večje vsebnosti rutina in kvercetina v moki tatarske ajde (Jiang et al. 2007; Qin et al. 2010), so bili izdelani kruhi iz tatarske ajde, z namenom, da bi preučevali učinke hidrotermične obde- lave zrnja, mlečnokislinske frementacije testa in vpliv peke na vsebnot rutina in kvercetina ter na antioksida- tivno aktivnost kruhov pripravljenih iz moke tatarske ajde in navadne ajde (Costantini et al. 2014; Lukšič et al. 2016a, 2016b). Eden od načinov priprave kruha vključuje mlečno- kislinsko fermentacijo, pri kateri se tvorita mlečna in ocetna kislina, ki vplivata na postopek priprave kislega kruha (Michalska et al. 2008). Raziskovalci so poroča- li, da mlečnokislinska fermentacija lahko vpliva na iz- boljšanje strukturnih in senzoričnih lastnosti kislega kruha in lahko podaljša obstojnost takega kruha (Gob- betti et al. 2014; Rizzello et al. 2016; Rinaldi et al. 2017; Ua Ar ak et al. 2017). V poskusih je bilo ugotovlje- no, da mlečnokislinka fermentacija lahko izboljša do- stopnost beljakovin, mineralov, skupno vsebnost pre- hranskih vlaknin, skupno vsebnost fenolnih snovi in antioksidativno ativnost kislih kruhov (Boskov Han - sen et al. 2002; Gandhi & Dey 2013; Costantini et al. 2014; Rizzello et al. 2016). Raziskovalci so poročali tudi, da je imel kisel kruh iz tatarske ajde manjšo vseb- nost ogljikovih hidratov, nižji glikemični indeks in manjšo energijsko vrednost, kot enaka količina moke tatarske ajde (Novotni et al. 2012; Costantini et al. 2014). V kislih kruhih so med procesom mlečnokislin- ske fermentacije nastajale tudi snovi, kot so alkoholi, al- dehidi, estri, hidrokarboni, ketoni, terpeni, furani, in fenoli (Boskov Hansen et al. 2002). Mikroorganizmi v osnovi za kislo testo prav tako lahko tvorijo nekatere nove prehranske komponente, kot so peptidi in drugi derivati aminokislin, in nekatere prebiotične polisahari- de (De Vos 2005). Ugotovljeno je bilo, da lahko protea- ze, ki jih v osnovi za kislo testo sproščajo kvasovke in encimi selekcioniranih laktobacilov, presnavljajo gluten v pšenični moki (Weiser et al. 2008). V raziskavah poročajo, da je, kot posledica različnih načinov toplotne obdelave v živilih, pripravljenih iz ta- tarske ajde in navadne ajde, prihajalo do zmanjšanja vsebnosti rutina in pretvorb le tega v kvercetin. Izmerili so tudi spremembe v vsebnosti drugih snovi z antioksi- dativnim učinkom (Vogrinčič et al. 2010; Zhang et al. 2010; Sakač et al. 2011). Cho & Lee (2015) sta poročala o tem, da na spremembe vsebnosti rutina in drugih an- tioksidantov hitro cvrtje rezancev ni vplivalo, med tem, ko je kuhanje povzročilo znatno zmanjšanje vsebnosti LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 82 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 rutina v rezancih, pripravljenih iz pšenične moke in iz z rutinom obogatenega izvečka otrobov tatarske ajde. Jambrec in sod. (2015) so ugotovili, da se je v polnozr- natih pšeničnih rezancih z dodatkom predhodno avto- klavirane (120 °C) moke navadne ajde, pretvorba rutina v kvercetin zmanjšala. Med kuho teh rezancev pa do pretvorbe rutina v kvercetin sploh ni prišlo. Izguba fe- nolnih snovi v rezancih z dodatkom ajdove moke je bila v območju kontrolnega vzorca (polnozrnatih pšeničnih rezancev). V poskusih, ki so jih opravili Qin in sod. (2013) so ugotovili, da je namakanje zrn tatarske ajde (40 °C, 12-14 h) vplivalo na zmanjšanje v zrnju prisotnega deleža škroba in rutina in vplivalo na povečanje vsebno- sti kvercetina, kaempferola, izokvercitrina, skupnih f la- vonoidov in fenolnih snovi. Po tem, ko so predhodno namočeno zrnje tatarske ajde obdelali še s paro (100 °C, 40-60 min), se je vsebnost skupnih flavonoidov in sku- pnih fenolnih snovi še naprej zmanjševala, med tem, ko se je vsebnost rutina v vzorcu zrnja povečala. Mogoče je, da je bil proces razgradnje rutina sprožen v procesu na- makanja zrnja tatarske ajde, proces obdelave s paro pa je sprožil ponovno spajanje rutina. Sensoy in sod. (2006) so predložili rezultate, ki nakazujejo da procesiranje (praženje) ajdove moke ni imelo vpliva na vsebnost sku- pnih fenolnih snovi v ajdovi moki. DPPH metoda dolo- čanja antioksidativne aktivnosti je sicer pokazala, da je prišlo po praženju ajdove moke (200°C, 10 min) do ra- hlega zmanjšanja antioksidativne aktivnosti moke ajde, praženje pri 170°C pa ni imelo vpliva na zmanjšanje an- tioksidativne aktivnosti. Do značilnega zmajšanja anti- oksidativne aktivnosti v moki tatarske ajde je prišlo, kot posledica različnih načinov toplotne obdelave moke, kot so praženje, segrevanja s paro in mikrovalovno segreva- nje (Zhang in sod. 2010). Izmerili so tudi manjše zmanj- šanje antioksidativne aktivnosti v moki navadne ajde, ki so jo pražili 10 min na 200 °C (Yasuda & Nakagawa 1994). Rezultati poskusov nakazujejo, da je optimizacija procesiranja ključna za ohranitev zdravju koristnih snovi v ajdovih izdelkih. Kruh je osnovno živilo za velik del svetovnega pre- bivalstva in lahko znatno prispeva k vnosu nekaterih hranil. Moka navadne ajde in posebno moka tatarske ajde vsebujeta nekatere fenolne snovi, kot sta rutin in kvercetin ter imata visoko antioksidativno aktivnost, zato sta primerni za pripravo kruhov z izboljšano pre- hransko vrednostjo in zdravju koristnimi lastnostmi. Poznavanje teh lastnosti moke je privedlo do raziskav v katerih so preučevali, kako tehnološki postopki priprave kruha iz navadne in tatarske ajde vplivajo na vsebnost, dostopnost in učinkovitost flavonoidov v ajdovih kru- hih. Preučevali so tudi razmerje med flavonoidoma, ru- tinom in kvercetinom v ajdi, in do kakšne mere se rutin pretvarja v kvercetin, kot posledica izpostavljenosti raz- ličnim tehnološkim postopkom priprave ajdovih kru- hov. Ugotovljeno je bilo, da se je v kruhu, pripravljenem iz moke tatarske ajde, vsebnost rutina zmanjševala, vsebnost kvercetina pa je naraščala in ostala stabilna med procesom priprave kruha (Vogrinčič et al. 2010). Kruhi, ki so vsebovali moko navadne ajde so vsebovali flavonoide, kot sta rutin in kvercetin in so imeli večjo antioksidativno aktivnost od pšeničnega kruha (Lin et al. 2009). Preučevani so bili tudi skupni učinki mlečnokislin- ske fermentacije in peke na vsebnost flavonoidov in an- tioksidativne lastnosti testa in kruhov iz moke navadne in tatarske ajde. Ugotovljeno je bilo, da je priprava kruha s postopkom mlečnokislinske fermentacije, iz moke navadne ajde in tatarske ajde možna brez dodatka pšenične moke ali glutena (Costantini et al. 2014; Lu- kšič et al. 2016a, 2016b). Kruhi pripravljeni iz 100 % moke tatarske ajde so imeli največjo vsebnost fenolov (53,3 mg GAE/g) in flavonoidov (16,8 mg RE/g), v kru- hih pripravljenih iz moke tatarske ajde z dodatkom 10% moke oljne kadulje (Salvia hispanica L.) pa so izmerili največjo antioksidativno aktivnost (32,0 mmol Fe 2+ E/g and 128,6 mmol GAE/g) v primerjavi s kruhi izdelani- mi iz 100% moke navadne ajde in pšenice in kruhi izde- lanimi iz moke navadne ajde in pšenice z dodatkom 10% moke oljne kadulje (Costantini et al. 2014). V na- sprotju s temi ugotovitvami, je bila v drugem poskusu, največja antioksidativna aktivnost izmerjena v kruhu iz moke navadne ajde v primerjavi s kruhom tatarske ajde. To je lahko posledica spajanja snovi z antioskidativnim učinkom, vključno snovi, ki lahko nastajajo med Mail- lardovo reakcijo, pod vplivom toplotne obdelave (Zhang et al. 2010; Lukšič et al. 2016a). O podobnih ugotovitvah so poročali tudi Vogrinčič in sod. (2010), ki so preučevali kruhe iz moke tatarske ajde in kruhe iz mešanice moke tatarske ajde in pšenice. V vseh vzorcih kruhov je med peko prišlo do zmanjšanja vsebnosti po- lifenolov. V mešanih kruhih iz moke tatarske ajde in pšenice in v kruhih iz moke tatarske ajde je med po- stopkom peke prišlo do zmanjšanja antioksidativne ak- tivnosti, ugotovljene z metodo DPPH (2,2-difenil-1-pi- krilhidrazil), med tem ko je do povečanja antioksidativ- ne aktivnosti, med postopkom peke, prišlo v kruhih iz 100 % pšenične moke. Lukšič in sod (2016a) so pripra- vili kisle kruhe iz moke navadne ajde in tatarske ajde, z namenom, da bi spremljali pretvorbe rutina in kverceti- na med postopkom mlečnokislinske fermentacije, pri- prave in peke kruha. Med mlečnokislinsko fermentaci- jo je prišlo do pretvorbe rutina v kvercetin. V kruhu pripravljenem iz moke tatarske ajde ni bilo rutina, bilo pa je 5,0 mg/g kvercetina. V kruhu iz moke navadne ajde niso izmerili ne vsebnosti rutina in ne kvercetina. Vogrinčič in sod. (2010) so poročali, da se je ob dodat- LUKŠIČ, GOLOB, MRAVIK & GERM: UV ABSORBING COMPOUNDS IN BUCKWHEAT PROTECT PLANTS 83 FOLIA BIOLOGICA ET GEOLOGICA 61/1 – 2020 ku vode testu iz moke tatarske ajde in kvasa, vsebnost rutina zmanjševala, vsebnost kvercetina pa je narašča- la. Vsebnost rutina se je še naprej zmanjševala med po- stopkom peke kruha, medtem ko je vsebnost kvercetina ostala stabilna. Po peki se je v kruhu iz 100% moke ta- tarske ajde ohranilo 0,47 mg/g rutina in 4,83 mg/g kver- cetina. Suzuki in sod. (2015) so pripravili kruh iz meša- nice moke tatarske ajde s sledovi rutinozidaze in pšeni- ce z 0,63 mg/g rutina, kar je približno 50 % delež ohra- njenega rutina v kruhu, v primerjavi z izvorno surovino (moko). Hidrotermična obdelava je postopek, ki vključuje segrevanje z vročo vodo ali paro, ki mu sledi ohlajanje in sušenje ajdovega zrnja z namenom, da bi pripravili oslu- ščeno ajdovo zrnje oziroma kašo. Gre za tradicionalen postopek, ki ga še vedno uporabljamo v Sloveniji, na Hr- vaškem, Poljskem, v Ukrajini in Rusiji (Kreft 2003). V raziskavi, ki so jo opravili Lukšič in sod. (2016b) smo preučevali učinek hidrotermične obdelave na dosto- pnost in izločanje flavonoidov iz škrobnih struktur. Zrnje tatarske ajde je bilo hidrotermično obdelano in zmleto v hidrotermično obdelano moko. V kontrolnem vzorcu, hidrotermično neobdelani moki, je bila večina kvercetina izločena že po 20 minutah ekstrakcije (8 mg/g DM (suhe mase)). V hidrotermično obdelani moki tatarske ajde je bilo po 20 min ekstrakcije, izločenega le 4 mg/g DM rutina, in 7 mg/g DM rutina po 8 h ekstrak- cije. Ti podatki nakazujejo, da je med postopkom hidro- termične obdelave, rutin postal vključen v druge struk- ture v moki in tako postal zaščiten pred pretvorbo v kvercetin med postopkom priprave kruha. Iz začetnih 7 mg/g DM v hidrotermično obdelani moki tatarske ajde, je kruh pripravljen iz te moke vseboval 2 mg/g DM ruti- na in 6 mg/g kvercetina. Za nobeno drugo metodo pri- prave kruha iz moke tatarske ajde ni bilo ugotovljeno, da bi se med postopkom priprave kruha ohranilo toliko rutina. Mnoge raziskave potrjujejo, da postopek mlečnoki- slinske fermentacije in toplotne obdelave vplivata na spremembe v vsebnosti in dostopnosti snovi z antioski- dativnimi lastnostimi v izdelkih iz ajde. Te spremembe se razlikujejo glede na vrsto ajde in lastnosti rastline, postopek priprave, metodo fermentacije in potopek to- plotne obdelave. Te informacije prispevajo k boljšemu razumevanju vpliva različnih metod priprave živil z an- tioksidativno aktivnostjo in obstojnosti rutina in kver- cetina v kislih kruhih in drugih prehranskih izdelkih. Te ugotovitve so pomembne tudi za pripravo živil z večjo vsebnostjo flavonoidov in izboljšano funkcijsko vrednostjo. ZAHV ALA Raziskovalna programa (št. P1-0212 »Biologija rastlin« in P3-0395 «Prehrana in javno zdravje» ) ter projekte L4- 7552, J4-5524 in L4-9305, slednjega s sofinansiranjem Ministrstva za kmetijstvo, gozdarstvo in prehrano, je sofinancirala Javna agencija za raziskovalno dejavnost Republike Slovenije iz državnega proračuna. Raziskavo je podpiral EUFORINNO 7 program EU za infrastruk- turo EU (RegPot št. 315982). Raziskave, ki smo jih opra- vili so prejele sredstva iz Evropske skupnosti v okviru projekta ITEM 26220220180: Building Research Centre «AgroBioTech». REFERENCES – LITERATURA Ačanski, M., Pastor, K., Psodorov , Đ., Vujić, Đ., Razmovski, R. & S. Kra vić, 2015: Determination of the presence of buckwheat flour in bread by the analysis of minor fatty acid methyl esters. Advanced technologies (Serbia) 4: 07–15. doi: https://10.5937/savteh1502086A. Barnes, P. W., Tobler, M. A., Keefover-Ring, K., Flint, S. D., Barkley, A. E., Ryel, R. J. & L. 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